CN115297964A - Magnetic sensor arrays for nucleic acid sequencing and methods of making and using the same - Google Patents

Abstract

Disclosed herein are devices for nucleic acid sequencing using magnetic labels (e.g., magnetic particles) and magnetic sensors. Methods of making and using these devices are also disclosed. The apparatus for nucleic acid sequencing comprises a plurality of magnetic sensors; a plurality of binding regions disposed above the plurality of magnetic sensors, the binding regions each for containing a fluid; and at least one conduit for detecting a characteristic of at least a first magnetic sensor of the plurality of magnetic sensors, the characteristic indicative of the presence or absence of one or more magnetic nanoparticles coupled to a first binding region associated with the first magnetic sensor.

Description

Magnetic sensor arrays for nucleic acid sequencing and methods of making and using the same

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application No. 62/987,831, filed 3/10/2020, and entitled "magnetic sensor array for nucleic acid sequencing and method of making and using same" (ROA-1001P-US/P35967-US attorney docket), and is incorporated herein by reference in its entirety.

Background

Synthetic sequencing (SBS) has become a successful commercially viable method for obtaining large amounts of DNA sequencing data. SBS involves the binding of primer hybridized template DNA, the introduction of deoxynucleoside triphosphates (dntps), and the detection of the introduced dntps.

Current sequencing systems use fluorescent signal detection. Four fluorescently labeled nucleotides were used to sequence millions of clusters in parallel. During successive cycles of DNA synthesis, DNA polymerase catalyzes the incorporation of fluorescently labeled dntps into the DNA template strand. During each cycle, a single labeled dNTP is added to the nucleic acid strand. The nucleotide tag serves as a "reversible terminator" for polymerization. After the dntps have been incorporated, the fluorochrome is identified by laser excitation and imaging, then enzymatically cleaved to allow the next round of introduction. During each cycle, bases were identified directly from signal intensity measurements.

The currently best technology sequencing systems that rely on fluorescence signal detection can provide up to 200 billion reads per run. However, achieving this performance requires large area flow cells, high precision free space imaging optics, and expensive high power lasers to generate sufficient fluorescent signals to successfully perform base detection.

Two general strategies have been to gradually increase SBS throughput (e.g., characterized by base reads per run). The first approach has been to scale out by increasing the size and number of flow cells in the sequencer. This approach increases the cost of reagents and the price of the sequencing system because it requires additional high power lasers and high precision nanopositioners.

The second approach involves inward scaling, where the size of individual DNA test sites is reduced, such that the number of DNA strands sequenced in a fixed-size flow cell is higher. This second approach is more attractive in reducing overall sequencing costs, as the additional cost is only related to achieving better imaging optics, while keeping the consumable costs unchanged. But a higher Numerical Aperture (NA) lens must be used to distinguish the signal from adjacent fluorophores. This approach has limitations because the Rayleigh (Rayleigh) standard sets the distance between resolvable light point sources to 0.61 λ/NA, that is, the minimum distance between two sequenced DNA strands cannot be reduced by more than about 400nm even in advanced optical imaging systems. A similar resolution limit applies to sequencing directly on top of the imaging array, on which the minimum pixel size obtained so far is less than 1 μm. The rayleigh standard currently represents a fundamental limitation for inward scaling of optical SBS systems. Overcoming these limitations may require super-resolution imaging techniques, which have not been implemented in highly multitasking systems. Therefore, at the present stage, the only viable way to increase the throughput of optical SBS sequencer is to build larger flow cells and more expensive optical scanning and imaging systems.

Therefore, there is still a need for improving SBS.

Drawings

The objects, features and advantages of the present invention will become apparent from the following description of certain embodiments thereof, taken in conjunction with the accompanying drawings, in which:

FIG. 1 illustrates a portion of a magnetic sensor according to some embodiments.

Fig. 2A and 2B illustrate the resistance of a Magnetoresistive (MR) sensor according to some embodiments.

FIG. 3A illustrates a concept of using a Spin Torque Oscillator (STO) sensor in accordance with some embodiments.

Fig. 3B shows an experimental reaction of an STO by a delay detection circuit when an AC magnetic field is applied across the STO according to some embodiments.

Fig. 3C and 3D illustrate the manner in which STO may be used as a nanoscale magnetic field detector according to some embodiments.

FIG. 4A is a top view of a portion of a sequencing device, according to some embodiments.

FIGS. 4B and 4C are cross-sectional views of portions of the sequencing device shown in FIG. 4A.

FIG. 4D is a block diagram showing components of the devices of FIGS. 4A, 4B, and 4C, according to some embodiments.

Fig. 5A and 5B illustrate two methods of selecting a magnetic sensor according to some embodiments.

FIG. 6 illustrates a method of manufacturing a sequencing device, according to some embodiments.

FIG. 7 illustrates a method for sequencing nucleic acids using a sequencing device, according to some embodiments.

FIG. 8 illustrates a method of using a sequencing device in which multiple nucleotide precursors are introduced substantially simultaneously according to some embodiments.

To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is contemplated that an assembly disclosed in one embodiment may be beneficially utilized on other embodiments without specific recitation. Moreover, the description of a component in the context of one drawing is applicable to other drawings that illustrate the component.

Detailed Description

Disclosed herein are devices for nucleic acid sequencing using magnetic labels (e.g., magnetic particles) and magnetic sensors. Methods of making and using these devices are also disclosed. For simplicity, some of the discussion below refers to sequencing DNA as an example. It is to be understood that the present disclosure applies generally to nucleic acid sequencing.

The inventors know that the resolution limits of fluorescence microscopy and complementary metal-oxide-semiconductor images (CMOS images), as used in prior art SBS, are not applicable to charge (e.g., silicon nanowire Field Effect Transistors (FETs)) or magnetic field sensors (e.g., spin valves, magnetic Tunnel Junctions (MTJs), spin Torque Oscillators (STO), etc.), where the size of the sensing elements is one order of magnitude smaller and also at a higher multitasking level than in the current best-technology SBS system. Magnetic field sensing in SBS is particularly attractive because DNA and sequencing reagents are non-magnetic, which can significantly improve the signal-to-noise ratio (SNR) compared to charge sensing schemes based on electron transport modulation in CMOS components. Furthermore, magnetic sensing does not require the incorporated base to be in direct contact with the junction. Miniaturized magnetic field sensors can be used to detect nanoscale magnetic nanoparticles for SBS.

SBS using an array of magnetic sensors can significantly increase throughput and reduce the cost of sequencing by providing additional inward scaling (e.g., about 100 times) while eliminating the need for high power lasers and high resolution optics in the sequencing system.

This document discloses SBS schemes that use magnetically labeled nucleotide precursors in conjunction with sequencing devices that include an array of magnetic sensing components (e.g., MTJs, STOs, spin valves, etc.). The device also includes one or more etched binding regions that allow a magnetic sensor to detect magnetic labels in magnetically labeled nucleotide precursors while protecting the magnetic sensor from damage (e.g., using a thin layer of insulator).

In the present disclosure, an apparatus for nucleic acid sequencing is disclosed, the apparatus comprising a plurality of magnetic sensors; a plurality of binding regions disposed above the plurality of magnetic sensors, the binding regions each for containing a fluid; and at least one conduit for detecting a characteristic of at least a first magnetic sensor of the plurality of magnetic sensors, the characteristic indicative of the presence or absence of one or more magnetic nanoparticles coupled to a first binding region associated with the first magnetic sensor. In some embodiments, the first magnetic sensor comprises a Magnetoresistive (MR) device. The MR device may include a pinned layer, a free layer, and a barrier layer configured between the pinned layer and the free layer. In some such embodiments, the magnetic moment of the pinned layer is about 90 degrees from the magnetic moment of the free layer in the absence of one or more magnetic nanoparticles coupled to the first binding region.

The first binding region can include a structure (e.g., a cavity or a ridge) configured to anchor the nucleic acid to the first binding region.

In some embodiments, the first magnetic sensor is substantially cylindrical or substantially cubical in shape. In some embodiments, the lateral dimension of the first magnetic sensor is between about 10 nanometers (nm) and about 1 micron.

The device may also include a sensing circuit coupled to the plurality of magnetic sensors via at least one pipeline. The sensing circuit may be configured to apply a current to the at least one line to detect a characteristic of the first magnetic sensor (e.g., a magnetic field, a resistance, a change in a magnetic field, a change in a resistance, a noise level, etc.). In some embodiments, the sensing circuitry includes a magnetically controlled oscillator, and the characteristic is a frequency of a signal associated with or generated by the magnetically controlled oscillator.

The device may have an insulating material (e.g., an oxide (e.g., silicon dioxide, aluminum oxide, etc.), a nitride (e.g., silicon nitride, etc.)) configured between the plurality of magnetic sensors and the plurality of bonding regions. The thickness of the insulating material between the top of the first magnetic sensor and the first binding region may, for example, be between about 3nm and about 20 nm.

In some embodiments, the at least one conduit comprises a first conduit disposed above a top surface of a first magnetic sensor, and the first binding region is located within a trench in the first conduit, the trench being above the top surface of the first magnetic sensor.

In some embodiments, the plurality of magnetic sensors are disposed in a rectangular array, and the at least one conduit comprises at least a first conduit and a second conduit, the first conduit being configured above the first magnetic sensor and the second conduit being configured below the first magnetic sensor. One or more union regions may be located within a groove in the first line. In some embodiments, the first pipeline is coupled to a row of the rectangular array and the second pipeline is coupled to a column of the rectangular array, or vice versa.

Also disclosed herein are methods of making devices for nucleic acid sequencing. In some embodiments, a method of manufacturing a nucleic acid sequencing device includes fabricating a first pipeline, fabricating a plurality of magnetic sensors, depositing an insulating material between the magnetic sensors, fabricating a plurality of additional pipelines, and creating a plurality of binding regions. In some embodiments, a bottom surface of each magnetic sensor is coupled to the first pipeline, and a top surface of each magnetic sensor is coupled to a respective one of the other pipelines.

Fabricating the first pipeline may include depositing a metal layer on the substrate (e.g., using physical vapor deposition, ion beam deposition, etc.), and patterning the metal layer into the first pipeline (e.g., using photolithography, milling, and/or etching).

In some embodiments, after fabricating the first pipeline and before fabricating the plurality of magnetic sensors, an insulating material is deposited over the first pipeline, the first pipeline is then exposed (e.g., using Chemical Mechanical Polishing (CMP)), and a plurality of magnetic sensors are fabricated on the exposed first pipeline.

Fabrication of a plurality of magnetic sensors may be by depositing a plurality of layers on a first pipeline, and patterning (e.g., using photolithography and/or etching) the plurality of layers to form a plurality of magnetic sensors, each having a predetermined shape (e.g., substantially cylindrical, substantially cubic, etc.). Depositing the plurality of layers may include depositing a first ferromagnetic layer, depositing a metal or insulating layer over the first ferromagnetic layer, and depositing a second ferromagnetic layer over the metal or insulating layer. The lateral dimension of each of the plurality of magnetic sensors may be, for example, between about 10nm and about 1 micron.

In some embodiments, the plurality of magnetic sensors are in a rectangular array, and the first pipeline corresponds to a row of the rectangular array, and the plurality of further pipelines each correspond to a column of the rectangular array, or vice versa.

In some embodiments, after depositing the insulating material between the magnetic sensors and before fabricating the plurality of additional lines, a chemical mechanical polishing step is performed to expose a top surface of each of the plurality of magnetic sensors.

In some embodiments, fabricating the plurality of additional lines includes depositing a metal layer, performing photolithography to define the plurality of additional lines, and removing a portion of the metal layer.

In some embodiments, creating a plurality of bonding regions includes applying a mask over the plurality of bonding regions, depositing (e.g., using atomic layer deposition) a metal layer over the mask, and picking the mask. After the mask is stripped, additional insulating material (e.g., an oxide such as silicon dioxide or the like or a nitride having a thickness between about 3nm and about 20 nm) may then be deposited over the plurality of additional lines and the plurality of bonding regions.

Also disclosed herein are methods of sequencing nucleic acids using the nucleic acid sequencing devices disclosed herein. In some embodiments, a method comprises (a) binding at least one nucleic acid strand to a first binding region, (b) adding an extendable primer and a nucleic acid polymerase to the first binding region in one or more additions, (c) adding a first nucleotide precursor to the first binding region, the first nucleotide precursor labeled with a first cleavable magnetic label, and (d) sequencing the nucleic acid strand. The first cleavable magnetic label can comprise a magnetic nanoparticle (e.g., a molecule, a superparamagnetic nanoparticle, a ferromagnetic nanoparticle, etc.). The first bonding area may be cleaned prior to step (c). After step (c), additional molecules of the nucleic acid polymerase may be added to the first binding region. During each repetition, steps (c) and (d) may be repeated with different nucleotide precursors, each magnetically labelled. The first nucleotide precursor can comprise one of dATP, dGTP, dCTP, dTTP, or an equivalent. The first and different nucleotide precursors can each be selected from the group consisting of magnetically labeled adenine, guanine, cytosine, thymine, or an equivalent thereof.

Sequencing nucleic acid strands can include using at least one line to detect a property of the first magnetic sensor that is indicative of the presence or absence of the first cleavable magnetic label. The characteristic may be, for example, a magnetic field or resistance, a frequency of a signal associated with or generated by the magnetically controlled oscillator, a noise level, or a change in a magnetic field or a change in resistance. The characteristic may result from a change in magnetic field or a change in resistance.

The method may further comprise the step of amplifying at least one nucleic acid strand. If performed, the amplification step may be performed before or after binding of the at least one nucleic acid strand to the first binding region. Due to the amplification, one or more amplicons may be bound to the first binding region.

In some embodiments, in response to a determination that the characteristic indicates the presence of one or more magnetic nanoparticles coupled to the first binding region, recording a complementary base of the first nucleotide precursor in a recording of a nucleic acid sequence of the nucleic acid strand.

In some embodiments, the first nucleotide precursor is not extendable by the nucleic acid polymerase, and the method further comprises, after detecting the property, removing the first cleavable magnetic label and making the first nucleotide precursor extendable by the nucleic acid polymerase. In some embodiments, the first nucleotide precursor is not extendable by the nucleic acid polymerase. The first nucleotide precursor may be rendered extendable by chemical cleavage.

After sequencing the nucleic acid strands, the cleavable magnetic label can be removed by enzymatic or chemical cleavage.

In some embodiments, the first cleavable magnetic label has a first magnetic property, and the method further comprises, in one or more additions, adding to the first binding region a second nucleotide precursor labeled with a second cleavable magnetic label having a second magnetic property. In some of these embodiments, the method further comprises, in one or more additions, adding to the first binding region a third nucleotide precursor labeled with a third cleavable magnetic label having a third magnetism, and a fourth nucleotide precursor labeled with a fourth cleavable magnetic label having a fourth magnetism.

In some embodiments, binding at least one nucleic acid strand to a first binding region comprises binding an adaptor to an end of a respective one of the at least one nucleic acid strand, and coupling an oligonucleotide to the first binding region, wherein the oligonucleotide is capable of hybridizing to the adaptor. In some embodiments, binding the at least one nucleic acid strand to the first binding region comprises covalently binding each of the at least one nucleic acid strands to the first binding region. In some embodiments, binding the at least one nucleic acid strand to the first binding region comprises immobilizing the at least one nucleic acid strand via irreversible passive adsorption or intermolecular affinity. In some embodiments, the first binding region comprises a cavity or a ridge, and binding the at least one nucleic acid strand to the first binding region comprises applying a hydrogel to the cavity or to the ridge.

In some embodiments, the nucleic acid polymerase is a type B polymerase lacking 3'-5' exonuclease activity. In some embodiments, the nucleic acid polymerase is a thermostable polymerase.

In some embodiments, using at least one line comprises applying a current to the at least one line.

Magnetic marker

The methods for sequencing nucleic acids described herein rely on the use of magnetically-labeled nucleotide precursors comprising cleavable magnetic labels. These cleavable magnetic labels may comprise, for example, magnetic nanoparticles such as, for example, molecules, superparamagnetic nanoparticles, or ferromagnetic particles. The magnetic labels may be nanoparticles having high magnetic anisotropy. Examples of nanoparticles having high magnetic anisotropy include, but are not limited to, fe ₃ O ₄ FePt, fePd, and CoPt. To facilitate chemical binding to nucleotides, particles can be synthesized and SiO used ₂ And (4) coating. See, e.g., mi Asi lam, l.fu, s.li and v.p. Delavade, "silica encapsulation and magnetic properties of FePt nanoparticles," journal of colloid and interface science, vol. 290, phase 2, p.2005, 10/15, p.444 to 449. Because of thisMagnetic labels of one size have permanent magnetic moments whose directions fluctuate randomly over a very short time scale, so some embodiments described further below rely on sensitive sensing schemes that detect fluctuations in the magnetic field caused by the presence of the magnetic labels.

There are many methods to bind magnetic labels to nucleotide precursors and to cleave the magnetic labels after incorporation of the nucleotide precursors. For example, the magnetic label may be bound to a base, in which case the magnetic label may be chemically cleaved. As another example, the magnetic label may be bound to a phosphate, in which case the magnetic label may be cleaved by a polymerase, or by cleaving a linker if bound via the linker.

In some embodiments, a magnetic label is attached to a nitrogenous base (A, C, T, G, or a derivative) of a nucleotide precursor. Upon incorporation of the nucleotide precursors and detection by a sequencing device (e.g., as described in further detail below), the magnetic labels are cleaved from the incorporated nucleotides.

In some embodiments, the magnetic label is bound via a cleavable linker. Cleavable linkers are known in the art and are described, for example, in U.S. patent nos. 7,057,026, 7,414,116, and their successors and modifications. In some embodiments, the magnetic label is bound to the 5-position in a pyrimidine or the 7-position in a purine via a linker comprising an allyl or azido group. In other embodiments, the linker comprises a disulfide bond, an indole, or a Ji Beier (Sieber) group. The linker may further contain one or more substituents selected from the group consisting of: alkyl (C) _1-6 ) Or alkoxy (C) _1-6 ) Nitro, cyano, fluoro or groups of similar nature. Briefly, the linker can be cleaved by a water-soluble phosphine or phosphine-based transition metal-containing catalyst. Other linkers and linker cleavage mechanisms are known in the art. For example, linker and acetal systems comprising trityl, p-alkoxybenzyl and p-alkoxybenzamide and a third butoxycarbonyl (Boc) group can be cleaved under acidic conditions by proton-releasing cleavage agentsAnd (5) solving. Thioacetals or other sulfur-containing linkers can be cleaved using sulfur-preferring metals such as nickel, silver, or mercury. Cleavage of the protecting group is also contemplated for the preparation of suitable linker molecules. Linkers containing ester and disulfide bonds can be cleaved under reducing conditions. Linkers containing Triisopropylsilane (TIPS) or tert-butyldimethylsilane (TBDMS) can be cleaved in the presence of F ions. Photocleavable linkers that are cleaved by wavelengths that do not affect the other components of the reaction mixture include linkers that comprise an O-nitrobenzyl group. The linker comprising a benzyloxycarbonyl group can be cleaved by a Pd-based catalyst.

In some embodiments, the nucleotide precursor comprises a label that binds to a polyphosphate moiety as described, for example, in U.S. patent nos. 7,405,281 and 8,058,031. Briefly, the nucleotide precursor comprises a nucleoside moiety and a chain of 3 or more phosphate groups, wherein one or more oxygen atoms are optionally substituted, for example with S. The label may be bound to the phosphate group of alpha, beta, gamma or higher (if present) either directly or via a linker. In some embodiments, the label is bound to the phosphate group via a non-covalent linker as described, for example, in U.S. patent No. 8,252,910. In some embodiments, the linker is a hydrocarbyl group selected from: substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocycloalkyl; see, for example, U.S. patent No. 8,367,813. The linker may further comprise a nucleic acid strand; see, for example, U.S. patent No. 9,464,107.

In embodiments where the magnetic label is attached to the phosphate group, the nucleotide precursor is incorporated into the nascent strand by a nucleic acid polymerase, which also cleaves and releases the detectable magnetic label. In some embodiments, the magnetic label is removed by cleaving the linker, e.g., as described in U.S. patent No. 9,587,275.

In some embodiments, the nucleotide precursor is a non-extendible "terminator" nucleotide, that is, a nucleotide whose 3' -terminus is blocked from adding the next nucleotide by a blocking "terminator" group. Blocking groups are reversible terminators that can be removed to continue the chain synthesis process as described herein. Binding removable blocking groups to nucleotides precursors are known in the art. See, for example, U.S. patent nos. 7,541,444 and 8,071,739, and their successors and amendments. Briefly, the blocking group may comprise an allyl group which may be cleaved by reaction with a metal-allyl complex in aqueous solution in the presence of a phosphine or nitrogen-phosphine ligand. Other examples of reversible terminator nucleotides used in synthetic sequencing include modified nucleotides described in: international application No. PCT/US2019/066670, filed on 16.12.2019 and entitled "3' -protected nucleotides", filed on 25.6.2020 and published as WO/2020/131759, which is incorporated herein by reference in its entirety for all purposes.

Magnetic sensor

Embodiments disclosed herein use magnetic sensors to detect the presence of a magnetic label coupled to a nucleotide precursor as described, for example, above.

FIG. 1 illustrates a portion of a magnetic sensor 105 according to some embodiments. The exemplary magnetic sensor 105 of FIG. 1 has a bottom surface 108 and a top surface 109 and includes three layers, e.g., two ferromagnetic layers 106A, 106B separated by a nonmagnetic spacer layer 107. The nonmagnetic spacer layer 107 may be, for example, a metallic material such as, for example, copper or silver, in which case the structure is referred to as a Spin Valve (SV), or it may be an insulator such as, for example, aluminum oxide or magnesium oxide, in which case the structure is referred to as a Magnetic Tunnel Junction (MTJ). Suitable materials for use in the ferromagnetic layers 106A, 106B include, for example, alloys of Co, ni, and Fe (sometimes mixed with other elements). In some embodiments, the ferromagnetic layers 106A, 106B are engineered to have their magnetic moments oriented in or perpendicular to the plane of the film. Additional materials may be deposited below and above the three layers 106A, 106B, and 107 shown in fig. 1 for purposes such as interface smoothing, texturing, and protection from processing for the patterning device 100, but with the active area of the magnetic sensor 105 in these three layer structures. Thus, a component in contact with the magnetic sensor 105 may be in contact with one of the three layers 106A, 106B, or 107, or it may be in contact with another portion of the magnetic sensor 105.

As shown in fig. 2A and 2B, the resistance of the MR sensor is proportional to 1-cos (θ), where θ is the angle between the moments of the two ferromagnetic layers 106A, 106B shown in fig. 1. To maximize the signal generated by the magnetic field and provide a linear reaction of the magnetic sensor 105 to the applied magnetic field, the magnetic sensor 105 may be designed such that the moments of the two ferromagnetic layers 106A, 106B are oriented pi/2 or 90 degrees with respect to each other in the absence of a magnetic field. Such orientation can be achieved by a number of methods known in the art. For example, one solution is to use an antiferromagnet to "pin" the magnetization direction of one of the ferromagnetic layers (106A or 106B, referred to as "FM 1") by an effect called exchange bias voltage, and then coat the sensor with a bilayer having an insulating layer and a permanent magnet. The insulating layer avoids electrical shorting of the magnetic sensor 105, and the permanent magnet supplies a "hard bias voltage" magnetic field perpendicular to the FM1 pinning direction, which will then rotate the second ferromagnetic body (106B or 106A, referred to as "FM 2") and produce the desired configuration. The magnetic field parallel to FM1 then rotates FM2 around this 90 degree configuration, and the change in resistance generates a voltage signal that can be calibrated to measure the magnetic field acting on the magnetic sensor 105. In this way, the magnetic sensor 105 acts as a magnetic field to voltage converter.

Note that while the example discussed immediately above describes the use of ferromagnets with moments oriented at 90 degrees relative to each other in the film plane, a perpendicular configuration may alternatively be achieved by orienting the moment of one of the ferromagnetic layers 106A, 106B out of the film plane, which may be done using a method known as Perpendicular Magnetic Anisotropy (PMA).

In some embodiments, the magnetic sensor 105 uses a quantum mechanical effect known as spin transfer torque. In these devices, current through one ferromagnetic layer 106A (or 106B) in the SV or MTJ preferentially allows electrons with spins parallel to the layer torque to pass through, while electrons with anti-parallel spins are more likely to be reflected. In this way, the current becomes spin polarized with more electrons of one spin type than the other. This spin-polarized current then interacts with the second ferromagnetic layer 106B (or 106A) to exert a torque on the torque of the layer. The torque may otherwise cause a moment of the second ferromagnetic layer 106B (or 106A) to precess about an effective magnetic field acting on the ferromagnetic body, or it may cause the moment to reversibly switch between two directions defined by uniaxial anisotropy induced in the system. The frequency of the resulting Spin Torque Oscillator (STO) can be adjusted by varying the magnetic field acting on it. Thus, it has the ability to act as a magnetic field to frequency (or phase) converter, as shown in FIG. 3A, which sets forth the concept of using an STO sensor. FIG. 3B shows the experimental reaction of an STO through a delay detection circuit when an AC magnetic field having a frequency of 1GHz and a peak-to-peak amplitude of 5mT is applied across the STO. This result and those shown in fig. 3C and 3D for short nanosecond field pulses illustrate the manner in which these oscillators can be used as nanoscale magnetic field detectors. For more details, see t, kawasaki, h, rattan, k, gongteng, t, ocean, k, water island, and r, zoxta, "delayed detection of a spin torque oscillator frequency modulation signal under a nanosecond pulsed magnetic field," journal of applied physics ", volume 111, 07C908 (2012).

Apparatus for nucleic acid sequencing

FIGS. 4A, 4B, and 4C illustrate portions of an apparatus 100 for nucleic acid sequencing according to some embodiments. Fig. 4A is a top view of the device. Fig. 4B is a cross-sectional view at a position indicated by a long dashed line labeled "4B" in fig. 4A, and fig. 4C is a cross-sectional view at a position indicated by a long dashed line labeled "4C" in fig. 4A. As shown in fig. 4A, the device 100 includes an array 110 of magnetic sensors, the array including a plurality of magnetic sensors 105, sixteen magnetic sensors 105 being shown in the array 110. To avoid obscuring the drawings, only seven magnetic sensors 105, namely magnetic sensors 105A, 105B, 105C, 105D, 105E, 105F and 105G, are labeled in fig. 4A. (for simplicity, this document refers generally to the magnetic sensors by reference numeral 105. Individual magnetic sensors are given by reference numeral 105 followed by a letter). The device 100 also includes at least one line 120, and for at least some of the magnetic sensors 105, a binding region 115 for each of those magnetic sensors 105, both of which are discussed in further detail below.

Portions of the magnetic sensors 105 and the pipeline 120 within the magnetic sensor array 110 are illustrated with dashed lines to indicate that they may not be visible in the top view of the device 100. As explained in further detail below, the magnetic sensor 105 is embedded in the device 100 and protected from the contents (e.g., insulation) of the binding region 115. Accordingly, it should be appreciated that various components set forth herein (e.g., the pipeline 120, the magnetic sensor 105, etc.) may not be visible in the physical instantiation of the device 100 (e.g., they may be embedded in or covered by a protective material such as an insulator).

In some embodiments, the magnetic sensors 105 in the magnetic sensor array 110 are each thin film devices that use the magneto-resistive (MR) effect to detect magnetic labels in the associated binding region 115, described in further detail below. As described in more detail below, each magnetic sensor 105 may operate as a potentiometer, wherein the resistance varies as the strength and/or direction of the sensing magnetic field changes.

The exemplary magnetic sensor array 110 in the exemplary embodiment of FIG. 4A is a rectangular array in which the magnetic sensors 105 are disposed in rows and columns. In other words, the plurality of magnetic sensors 105 of the magnetic sensor array 110 are disposed in a rectangular grid pattern. It should be appreciated that disposing the magnetic sensors 105 in a grid pattern as shown in fig. 4A is one of many possible arrangements. One of ordinary skill in the art will appreciate that other placements of the magnetic sensor 105 are possible and within the scope of the invention herein.

Referring now to fig. 4B and 4C, in conjunction with fig. 4A, each magnetic sensor 105 illustrated in the exemplary embodiment of the device 100 has a cylindrical shape. However, it should be understood that, in general, the magnetic sensor 105 may have any suitable shape. For example, the magnetic sensor 105 may be cuboidal in three dimensions. Furthermore, different magnetic sensors 105 may have different shapes (e.g., some may be cuboidal and others may be cylindrical, etc.).

As shown in the exemplary embodiments of fig. 4A, 4B, and 4C, the bonding region 115 is disposed above each magnetic sensor 105. For example, the binding region 115A is above the magnetic sensor 105A; the binding region 115B is above the magnetic sensor 105B; the binding region 115C is above the magnetic sensor 105C; the binding region 115D is above the magnetic sensor 105D; the binding region 115E is above the magnetic sensor 105E; the binding region 115F is above the magnetic sensor 105F; and the binding region 115G is above the magnetic sensor 105G. The other unlabeled nine magnetic sensors 105 shown in fig. 4A are also each disposed below a respective binding region 115 (also not labeled in fig. 4A).

The bonding region 115 contains a fluid. The magnetic sensor 105 may detect magnetic labels (e.g., nanoparticles) in the binding region 115. Thus, in some embodiments, the surface 116 of each binding region 115 has properties and characteristics that protect the magnetic sensor 105 from any fluid in the binding region 115, while still allowing the magnetic sensor 105 to detect magnetic labels within the binding region 115. The material of the surface 116 (and possibly the remainder of the bonding region 115) may be or include an insulator. For example, in some embodiments, the surface 116 comprises polypropylene, gold, glass, or silicon. It is to be understood that the surface 116 can be an exposed surface of a multi-layer structure configured over the pipeline(s) 120 located over the magnetic sensor 105. For example, in embodiments where the surface 116 includes a conductor (e.g., gold), a layer of insulating material may be used to separate the conductor from the pipeline 120 above the magnetic sensor 105. (see FIGS. 4B and 4C). The thickness of the surface 116 may be selected to distance the magnetic sensor 105 from the binding region 115 such that the magnetic sensor 105 is able to detect magnetic labels within the binding region 115. In some embodiments, the surface 116 is about 3 to 20nm thick such that the sensing layer of the magnetic sensor 105 (described further below) is about 5nm to about 40nm from the magnetic labels in its respective binding region 115.

In some embodiments, the surface 116 of the binding region 115 has a structure (or structures) configured to anchor nucleic acids to the surface 116. For example, the structure (or structures) may comprise cavities or ridges. Further, in some embodiments, the surface 116 has properties that facilitate nucleic acid amplification. For example, the apparatus 100 can facilitate bridge amplification to facilitate the generation of clonal clusters of a single nucleic acid strand within each of the binding regions 115.

The shape of each bonding region 115 shown in the exemplary embodiments of fig. 4A, 4B, and 4C is cubical (e.g., as shown in fig. 4A, each bonding region 115 has a square shape when viewed from the top and is then rectangular when viewed in cross-section), although it is understood that the bonding regions 115 may have other shapes (e.g., circular, elliptical, octagonal, etc.). For example, the shape of the binding region may be similar or identical to the shape of the magnetic sensor 105 (e.g., if the magnetic sensor 105 is cylindrical in three dimensions, the binding region 115 may also be cylindrical with a radius larger, smaller, or the same size as the radius of the magnetic sensor 105; if the magnetic sensor 105 is cuboidal in three dimensions, the binding region 115 may also be cuboidal with a surface 116 larger, smaller, or the same size as the top of the magnetic sensor 105, etc.). Moreover, the different bonding areas 115 and the different surfaces 116 may have different shapes (e.g., some surfaces 116 may be circular, some may be rectangular, some may be square, etc.). Additionally, although fig. 4B and 4C show the bonded area 115 having vertical sides, the sides need not be vertical. In general, the binding region 115 and its surface 116 may have any shape and characteristics that facilitate detection of magnetic nanoparticles in the binding region 115 by the magnetic sensor 105.

In some embodiments, such as the exemplary embodiment set forth in fig. 4A, 4B, and 4C, a plurality of magnetic sensors 105 are each coupled to at least one pipeline 120. (for simplicity, this document generally indicates pipelines by reference number 120. Individual pipelines are given by reference number 120 followed by letters). In the exemplary embodiment shown in fig. 4A, 4B, and 4C, each magnetic sensor 105 in the magnetic sensor array 110 is coupled to two pipelines 120. For example, magnetic sensor 105A is coupled to lines 120A and 120H; magnetic sensor 105B is coupled to lines 120B and 120H; magnetic sensor 105C is coupled to lines 120C and 120H; magnetic sensor 105D is coupled to lines 120D and 120H; magnetic sensor 105E is coupled to lines 120D and 120E; magnetic sensor 105F is coupled to lines 120D and 120F; and the magnetic sensor 105G is coupled to pipelines 120D and 120G. In the exemplary embodiment of fig. 4A, 4B, and 4C, the pipelines 120A, 120B, 120C, and 120D are shown below the magnetic sensor 105, and the pipelines 120E, 120F, 120G, and 120H are shown above the magnetic sensor 105.

FIG. 4B shows that magnetic sensor 105E is associated with lines 120D and 120E, magnetic sensor 105F is associated with lines 120D and 120F, magnetic sensor 105G is associated with lines 120D and 120G, and magnetic sensor 105D is associated with lines 120D and 120H. FIG. 4C shows magnetic sensor 105D associated with lines 120D and 120H, magnetic sensor 105C associated with lines 120C and 120H, magnetic sensor 105B associated with lines 120B and 120H, and magnetic sensor 105A associated with lines 120A and 120H.

The pipelines 120 of the exemplary embodiments of fig. 4A, 4B, and 4C each identify a row or column of the magnetic sensor array 110. For example, the pipelines 120A, 120B, 120C, and 120D each identify a different row of the magnetic sensor array 110, and the pipelines 120E, 120F, 120G, and 120H each identify a different column of the magnetic sensor array 110. As shown in fig. 4B, each of the lines 120E, 120F, 120G, and 120H is in cross-sectional contact with one of the magnetic sensors 105 (i.e., line 120E is in contact with the top of the magnetic sensor 105E, line 120F is in contact with the top of the magnetic sensor 105F, line 120G is in contact with the top of the magnetic sensor 105G, and line 120H is in contact with the top of the magnetic sensor 105D), and line 120D is in contact with the bottom of each of the magnetic sensors 105E, 105F, 105G, and 105D. Also, and as shown in fig. 4C, each of the lines 120A, 120B, 120C, and 120D is in cross-sectional contact with the bottom of one of the magnetic sensors 105 (i.e., line 120A is in contact with the bottom of magnetic sensor 105A, line 120B is in contact with the bottom of magnetic sensor 105B, line 120C is in contact with the bottom of magnetic sensor 105C, and line 120D is in contact with the bottom of magnetic sensor 105D), and line 120H is in contact with the top of each of the magnetic sensors 105D, 105C, 105B, and 105A.

In some embodiments, some or all of the bonding regions 115 are located within a trench in the pipeline 120 that passes over the magnetic sensor 105. For example, as shown in fig. 4C, the line 120H is thinner above the magnetic sensors 105 than it is between the magnetic sensors 105. For example, the pipeline 120H has a first thickness over the magnetic sensor 105D, a second, greater thickness between the magnetic sensors 105D and 105C, and a first thickness over the magnetic sensor 105C.

For simplicity of illustration, fig. 4A, 4B, and 4C illustrate an exemplary device 100 having only sixteen magnetic sensors 105, only sixteen corresponding binding regions 115, and eight pipelines 120 in the magnetic sensor array 110. It should be appreciated that the magnetic sensor array 110 of the device 100 may have fewer or more magnetic sensors 105, and it may have more or less binding regions 115, and it may have more or less pipelines 120. In general, any configuration of the magnetic sensor 105 and the binding region 115 that allows the magnetic sensor 105 to detect magnetic labels in the binding region 115 may be used. Likewise, any configuration of one or more lines 120 that allows for determining whether the magnetic sensor 105 has one or more magnetic labels sensed may be used.

FIG. 4D is a block diagram showing components of the apparatus 100 according to some embodiments. As shown, the device 100 includes an array of magnetic sensors 110 coupled by a pipeline 120 to a sensing circuit 130. In operation, the sensing circuit 130 can apply a current to the conduit 120 to detect a characteristic of at least one of the plurality of magnetic sensors 105 in the magnetic sensor array 110, wherein the characteristic is indicative of the presence or absence of magnetically labeled nucleotide precursors in the binding region 115. For example, in some embodiments, the property is a magnetic field or resistance, or a change in a magnetic field or a change in resistance. In some embodiments, the characteristic is a noise level. In some embodiments, the magnetic sensor includes a magnetically controlled oscillator and the characteristic is a frequency of a signal associated with or generated by the magnetically controlled oscillator.

In some embodiments, the sensing circuit 130 detects deviations or fluctuations in the magnetic environment of some or all of the magnetic sensors 105 in the magnetic sensor array 110. For example, a magnetic sensor 105 of the MR type in the absence of a magnetic marker should have relatively less noise above a certain frequency than a magnetic sensor 105 in the presence of a magnetic marker, since fluctuations in the magnetic field from the magnetic marker will cause fluctuations in the sensed ferromagnetic torque. These fluctuations may be measured using heterodyne detection (e.g., by measuring noise power density) or by directly measuring the voltage of the magnetic sensor 105 and evaluated using a comparator circuit for comparison with a virtual sensor component that does not sense the binding region 115. Where the magnetic sensor 105 includes an STO component, the fluctuating magnetic field from the magnetic marker will cause a phase jump of the magnetic sensor due to the instantaneous change in frequency, which can be detected using a phase detection circuit. Another option is to design the STO to oscillate only within a small magnetic field range so that the presence of the magnetic label will turn off the oscillation. It should be understood that the examples provided above are exemplary only. Other detection methods are contemplated and are within the scope of the present invention.

In some embodiments, the magnetic sensor array 110 includes a selector component that reduces the likelihood of "sneak" currents that may be transmitted through adjacent components and degrade the performance of the magnetic sensor array 110. Fig. 5A and 5B illustrate two methods according to some embodiments. In fig. 5A, CMOS transistors are coupled in series with the magnetic sensor 105. For more details on the configuration shown in fig. 5A, see b.n. engel, j. Ackman, b. Buchner, r.w. davf, m. Dehira, m. Du Lanm, g. Glenkville, j. Jian Siji, s.v. Pi Ai tambam, n.d. rizo, j.m. slaalt, k. Smith, j.j. Sun, and s. Terani, "a 4-trigger MRAM based on new bit and switching method", mb IEEE magnetic exchange, volume 41, 132 (2005).

In fig. 5B, diodes or diode-like components are deposited with the magnetic films and then placed within a "cross-point" architecture, where CMOS transistors at the periphery of the magnetic sensor array 110 turn on individual pipelines 120 (e.g., word lines and bit lines) to address individual magnetic sensors 105 in the array. The use of CMOS select transistors can be made simpler due to the popularity of foundries available for the fabrication of front ends (e.g., all nanofabrication building CMOS transistors and underlying circuitry), but the type of current required for operation can require cross-point design to ultimately achieve the required density for the magnetic sensor array 110. Additional details regarding the configuration shown in fig. 5B can be found in c. Cabot, a. Feldt and f.n. Fan Daoer, "the rise of spintronics in data storage," nature materials, volume 6, 813 (2007).

In some embodiments, use of device 100 allows for amplification of nucleic acids, such as, for example, using bridge amplification (discussed further below). The distance between individual strands in a clonal cluster generated by an amplification procedure, such as described in more detail below, can be estimated to select the size and density of the magnetic sensors 105 in the magnetic sensor array 110. For example, to estimate the distance, the skilled person can consider both the contour length (e.g. the length of the stretched strand of DNA) and the persistence length (e.g. the average length after strand bending during a bridge amplification procedure) of a 200 Base Pair (BP) double-stranded DNA, to choose because the average strand length for many nucleic acid sequencing amplifications is 200BP, and double-stranded DNA is less flexible than single-stranded DNA and therefore provides an upper limit. The mean profile length is about 65nm, and the persistence length is about 35nm (see, e.g., s. Boolean et al, "determination of persistence length of double-stranded DNA using dark-field tethered particle motion", journal of chemico-physics (2009) 130. Because DNA bends during the bridging amplification process, the average distance between amplified clones should be between the contour length and the persistence length. Thus, the distance can be estimated to be about 40nm. Assuming that between tens and hundreds of copy chains may be required to sequence each initially bound target chain from a signal-to-noise ratio (SNR) perspective, the magnetic sensor 105 may have a size on the order of, for example, about 10nm to about 1 μm. It will be appreciated that because the sequencing uses magnetic nanoparticles rather than fluorescence, the spacing between adjacent magnetic sensors 105 can be much smaller than that required by the optical system, which is limited by diffraction effects. For example, in embodiments of the device 100 disclosed herein, adjacent magnetic sensors 105 may be between about 20nm and about 30nm apart.

Method of manufacturing a sequencer device

In some embodiments, the apparatus 100 is fabricated using a photolithography process and thin film deposition.

Fig. 6 illustrates a method 150 of manufacturing the device 100 according to some embodiments. At 152, the method begins. At 154, at least one pipeline 120 (e.g., first pipeline 120) is fabricated on a substrate, for example, by depositing a metal layer on the substrate, and patterning the metal layer into the at least one pipeline 120. The metal layer may be deposited, for example, using Physical Vapor Deposition (PVD) or Ion Beam Deposition (IBD). Patterning the metal layer into the at least one line 120 can be performed using photolithography, milling, and/or etching.

Optionally, at 156, an insulating material may be deposited over the at least one pipeline 120, and then, also optionally, at 158, the at least one pipeline 120 may be exposed. For example, the at least one line 120 may be exposed using Chemical Mechanical Polishing (CMP).

At 160, a plurality of magnetic sensors 105 (e.g., magnetic sensor array 110) are fabricated on at least one pipeline 120. The plurality of magnetic sensors 105 may be fabricated, for example, by depositing a plurality of layers on the at least one pipeline 120, and then patterning the plurality of layers to form the plurality of magnetic sensors 105. The multiple layers may be deposited using any suitable technique. For example, the plurality of layers may be deposited by: a first ferromagnetic layer (e.g., layer 106B shown in fig. 1) is deposited, a metal or insulating layer (e.g., layer 107 shown in fig. 1) is deposited over the first ferromagnetic layer, and a second ferromagnetic layer (e.g., layer 106A shown in fig. 1) is deposited over the metal or insulating layer. Patterning the plurality of layers to form the plurality of magnetic sensors 105 may be performed using any suitable technique, such as, for example, photolithography or etching.

In some embodiments, each magnetic sensor 105 of the magnetic sensor array 110 has a bottom surface 108 and a top surface 109. (see, e.g., FIG. 1). The bottom surface 108 is coupled to one of the at least one line 120 (e.g., the bottom surface 108 is coupled to a first line 120). In some embodiments, the bottom surface 108 of each magnetic sensor 105 is in contact with one of the at least one tubing lines 120 (e.g., the first tubing line 120).

In some embodiments, the plurality of magnetic sensors 105 each have a predetermined shape that may be the same for all of the plurality of magnetic sensors 105 or may be different for two or more of the magnetic sensors 105. The predetermined shape may be any suitable shape, including, for example, substantially cylindrical or substantially cubical. The lateral dimension of each of the plurality of magnetic sensors 105 may be, for example, between about 10nm and about 1 μm. As used herein, the term "lateral dimension" refers to a dimension in the x-y plane shown in fig. 4A, for example, when the device 100 is viewed from the top. For example, when the magnetic sensor 105 is cylindrical, the lateral dimension is the diameter of the top surface 109 of the cylinder. As another example, when the magnetic sensor 105 is cubical, its lateral dimensions include the dimensions of its top surface (e.g., the length, width, or diagonal dimensions of its top surface 109).

Referring again to the method embodiment of FIG. 6, at 162, an insulating material (e.g., a dielectric material) is deposited between the magnetic sensors 105 of the magnetic sensor array 110. The insulating material may be any suitable material such as, for example, an oxide or nitride. For example, the insulating material may comprise silicon dioxide (SiO) ₂ ) Alumina (Al) ₂ O ₃ ) Or silicon nitride (Si) ₃ N ₄ )。

Optionally, at 164, a chemical mechanical polishing step may be performed to expose the top surface 109 of each of the plurality of magnetic sensors 105.

At 166, at least one additional line 120 is fabricated using any suitable technique. The at least one further line 120 may be manufactured, for example, by depositing a metal layer, performing photolithography to define the at least one further line 120, and removing a portion of the metal layer, leaving the at least one further pipeline 120.

In some embodiments, the at least one additional line 120 is each coupled to the top surface 109 of at least one magnetic sensor 105 in the array of magnetic sensors 110. In some embodiments, the top surface 109 of each magnetic sensor 105 is in contact with the same conduit 120. In some embodiments, the bottom surface 108 of the magnetic sensor 105 is in contact with the first tubing 120A and the top surface 109 of the magnetic sensor 105 is in contact with the second tubing 120B.

In some embodiments, the plurality of magnetic sensors 105 is in a rectangular magnetic sensor array 110. In these embodiments, at least one pipeline 120 (e.g., a first or bottom pipeline 120) may correspond to one or more rows of the rectangular array and the at least one other pipeline 120 (e.g., a second or top pipeline 120) may correspond to one or more columns of the rectangular array, or vice versa.

At 168, a plurality of bonding regions 115 are created using any suitable technique. For example, the plurality of binding regions 115 may be generated by: applying a mask over regions corresponding to the plurality of bonding regions 115, depositing a metal layer over the mask, and picking the mask. For example, photolithography can be performed to define a mask where a window in the polymer overlaps the top line 120, except directly above the magnetic sensor 105. Subsequent metal deposition and stripping can then be performed to thicken the top line 120 away from the magnetic sensors 105, which creates shallow trenches above each magnetic sensor 105 and reduces the resistance of the top line 120 to improve noise performance. These shallow trenches may define the bonding region 115.

Thus, in some embodiments, the plurality of binding regions 115 is created by: a trench is fabricated in the top pipeline 120 at a location corresponding to the top of the magnetic sensor 105, and then an insulating material is deposited over the trench. For example, in embodiments where the plurality of magnetic sensors 105 are disposed in a rectangular array 110 with some pipelines 120 (bottom pipelines 120) below the magnetic sensors 105 and other pipelines 120 (top pipelines 120) above the magnetic sensors 105, trenches may be etched in each of the top pipelines 120 at locations where the top pipelines pass over the magnetic sensors 105. The bonding region 115 is then defined by a trench above the magnetic sensor 105 (e.g., as shown in fig. 4C).

In some embodiments, after the generation of the bonding regions 115 (e.g., after picking up the mask and/or generating the trenches described above), a further insulating material (e.g., an oxide, such as SiO, is deposited (e.g., using atomic layer deposition)) is deposited (e.g., over the plurality of further pipelines 120 and the plurality of bonding regions 115 ₂ Or nitride). The thickness of the further insulating material may, for example, be between about 3nm and about 20 nm. For this purpose, any suitable insulating material that electrically isolates the magnetic sensor 105 from the magnetic labels in the binding region 115 and protects the magnetic sensor 105 from fluids that are expected to be added to the binding region 115 may be used. For example, the additional insulating material may comprise silicon dioxide (SiO) ₂ ) Aluminum oxide (AlO) _x ) Or silicon nitride (SiN), or the like.

At 170, method 150 ends.

Sequencing method

In some embodiments, the nucleic acids are sequenced using immobilized nucleic acid strands (possibly in a clone cluster) that are tethered to the vicinity of the magnetic sensors 105 of the magnetic sensor array 110 of the device 100. Four types of reversible terminator bases (RT-bases) can then be added simultaneously or one at a time, and unincorporated nucleotides washed away. The magnetic label along with the terminal 3' blocker can then be chemically removed from the nucleic acid strand before the next sequencing cycle is initiated.

The nucleic acid strand may be prepared in any suitable manner. For example, the nucleic acid strands can be prepared by randomly fragmenting a nucleic acid sample followed by 5 'and 3' adaptor ligation. These strands of the nucleic acid can then be captured on oligonucleotides bound or attached to the surface 116 of at least some of the binding regions 115. Linear or exponential amplification (including bridge amplification) can be used to amplify the strands prior to sequencing.

Bridging amplification and other amplification techniques are well known in the art and may be used with the device 100 according to some embodiments. To begin bridging amplification, the nucleic acids to be sequenced can be attached to a substrate using, for example, adaptor strands immobilized in a hydrogel, for example. A polymerase, primer, and nucleotide precursor can then be introduced into the binding region 115 to generate a double-stranded nucleic acid from the single target strand. The double strand may then be denatured, which separates the double-sided nucleic acid strand into two single strands that are complementary to each other. As shown herein, bridge formation involves a chemical process that causes the single strand to fold and attach to a complementary adaptor strand immobilized on a substrate. Again, a polymerase, primers, and nucleotide precursors are introduced into the binding region 115 to convert the individual single-stranded "bridges" into double side chains. After this step, the double strand is denatured to produce complementary single strands, one being the original "forward" strand and the other being the replicated "reverse" strand. After repeating these steps multiple times, clonal clusters are formed with both forward and reverse ditto. One of the two clusters (e.g., the reverse strand) can then be cleaved from the binding region 115, and the remaining clusters (e.g., the forward strand) then sequenced.

The use of an amplification program in conjunction with the apparatus 100 for nucleic acid sequencing may improve the SNR of the sequencing process and thereby improve the accuracy of the sequencing. The SNR improvement results due to the presence of many copies of the same nucleic acid strand to be sequenced within the binding region 115 allow for the incorporation of larger amounts of magnetically labeled nucleotide precursors within the binding region 115. Incorporating a larger amount of magnetically labeled nucleotide precursors further increases the likelihood that the magnetic sensor 105 associated with the binding region 115 will detect the presence of the magnetic label within the binding region 115. Thus, having a larger number of copies of the strand to be sequenced reduces the likelihood that the magnetic sensor 105 will miss incorporation of magnetically labeled nucleotide precursors and thereby generate sequencing errors.

To sequence nucleic acid strands, magnetically-labeled nucleotide precursors can be introduced one at a time or all at once, as described below.

In some embodiments, the magnetically-labeled nucleotide precursors are introduced one at a time. In these embodiments, the same magnetic label may be used for all of the nucleotide precursors. It should be understood that, as used herein, the phrase "same magnetic marker" does not refer to the same physical instance of a single magnetic marker (that is, it does not represent a specific instance of repeated utilization of a physical marker); rather, it refers to multiple physical instantiations of a magnetic marker that all have the same characteristics or properties such that their individual instances are indistinguishable from one another. Conversely, the phrase "different magnetic labels" means that the magnetic labels (individually or as a group) have different characteristics or properties that allow them (whether individually or as a group) to be distinguished from other magnetic labels.

In some embodiments, the nucleic acid strands are extended one nucleotide at a time, and the magnetic sensor array 110 is used to recognize bound magnetically-labeled nucleotide precursors.

Fig. 7 is a flow diagram illustrating a method 200 of nucleic acid sequencing using the device 100 or another device that senses the presence or absence of magnetic labels using a magnetic sensor, according to some embodiments. At 202, the method begins. At 204, one or more nucleic acid strands are bound to the surface 116 of one or more binding regions 115 of the sequencing device 100, as described above. There are many ways to bind the one or more nucleic acid strands to the surface 116. For example, the nucleic acid strand may be bound to the surface 116 by binding an adapter to an end of the nucleic acid strand and coupling an oligonucleotide to the surface 116 of the binding region 115, wherein the oligonucleotide is complementary to the adapter. As another example, the nucleic acid strand may be bound to the surface 116 by covalently binding the nucleic acid strand to the surface 116. As yet another example, the nucleic acid strands may be bound to the surface 116 by immobilizing the nucleic acid strands via irreversible passive adsorption or intermolecular affinity. In some embodiments, the surface 116 includes cavities or ridges, as described above, and binding the nucleic acid strands to the proximal wall comprises applying a hydrogel to the cavities or to the ridges.

In optional step 206, the nucleic acid strand(s) may be amplified using any suitable method, such as, for example, by using Polymerase Chain Reaction (PCR) or linear amplification.

At 208, an extendable primer is added to the binding region 115.

At 210, a nucleic acid polymerase is added to the binding region 115. The nucleic acid polymerase can be any suitable nucleic acid polymerase. Desirable characteristics of a nucleic acid polymerase (such as a DNA polymerase) for use in nucleic acid sequencing include one or more of: a fast association rate for the nucleic acid template and the nucleotide precursor, or a slow dissociation rate for the nucleic acid template and the nucleotide precursor (association and dissociation rates are kinetic properties of the nucleic acid polymerase under a defined set of reaction conditions); high fidelity, low or undetectable exonuclease activity, including low or undetectable 3'-5' exonuclease (proofreading) activity or low or undetectable 5'-3' exonuclease activity; efficient DNA strand displacement, high stability, high processability (including long read length), salt tolerance, and the ability to incorporate modified nucleotide precursors (including the precursors described herein).

Some examples of suitable polymerases include family B (type B) polymerases that lack 3'-5' exonuclease activity.

In some embodiments, the polymerase is a thermostable polymerase. Thermostable nucleic acid polymerases include Thermus aquaticus (Thermus aquaticus) Taq DNA polymerase, thermus sp Z05 polymerase, thermus flavus (Thermus flavus) polymerase, thermotoga maritima (Thermotoga maritima) polymerase such as TMA-25 and TMA-30 polymerase, tth DNA polymerase, pyrococcus furiosus (Pfu), pyrococcus wakakii (Pwo), thermotoga maritima (Thermotoga maritima) (Tma), and Thermococcus (Thermococcus Litoralis) (Tli or Vent), and the like.

In some embodiments, the polymerase lacks detectable 5'-3' exonuclease activity. Examples of DNA polymerases that substantially lack 5 'to 3' nuclease activity include the Klenow (Klenow) fragment of escherichia coli (e.coli) DNA polymerase I; thermus aquaticus DNA polymerase (Taq) lacking the N-terminal 235 amino acids ("Stoffel fragment"), see U.S. Pat. No. 5,616,494. Other examples include thermostable DNA polymerases with sufficient deletions (e.g., N-terminal deletions), mutations, or modifications to eliminate or not activate the domain responsible for 5'-3' nuclease activity. See, for example, U.S. patent No. 5,795,762.

In some embodiments, the polymerase lacks detectable 3'-5' exonuclease activity. Examples of DNA polymerases substantially lacking 3'-5' exonuclease activity include Taq polymerase and derivatives thereof, and any B-family (B-type) polymerase having a naturally occurring or engineered deletion of the proofreading domain.

In some embodiments, the polymerase has been modified or engineered to be capable of incorporating or enhancing incorporation of nucleotide analogs, such as 3' -modified nucleotides; see, for example, U.S. patent nos. 10,150,454, 9,677,057 and 9,273,352.

In some embodiments, the polymerase has been modified or engineered to incorporate or enhance the incorporation of nucleotide analogs, such as 5' -phosphate-modified nucleotides; see, for example, U.S. patent nos. 10,167,455 and 8,999,676. In some embodiments, the polymerases are phi 29-derived polymerases; see, for example, U.S. patent nos. 8,257,954 and 8,420,366. In some embodiments, these polymerases are phiCPV 4-derived polymerases; see, for example, U.S. patent publication No. US 20180245147.

In some embodiments, the polymerase is modified or engineered by choice to successfully incorporate the desired modified nucleotides or to incorporate nucleotides and nucleotide analogs with the desired precision and processability. Methods of selecting these modified polymerases are known in the art; see, for example, U.S. patent publication No. US20180312904A1 entitled "polymerase compositions and methods of making and using the same".

It should be understood that steps 208 and 210 may be combined or their order reversed.

Optionally, at 212, the binding region 115 can be washed and then a magnetically labeled nucleotide precursor is added at step 214.

At 228, magnetically labeled nucleotide precursors are selected for sequencing cycles. In some embodiments of the present invention, the, the magnetically labeled nucleotide precursor is selected from adenine, guanine, cytosine, thymine, or an equivalent thereof. In some embodiments, the magnetically-labeled nucleotide precursor comprises one of magnetically-labeled dATP, dGTP, dCTP, dTTP, or equivalent. The magnetically labelled nucleotide precursor may be labelled as a conventional, natural, unconventional or similar nucleotide. The terms "conventional" or "natural" when referring to nucleotide precursors refer to those naturally occurring (that is, with respect to DNA, these are dATP, dGTP, dCTP and dTTP). The term "unconventional" or "similar" when referring to a nucleotide precursor includes modifications or analogs of the conventional bases, sugar moieties or internucleotide linkages in the nucleotide precursor. For example, dITP, 7-deaza-dGTP, 7-deaza-dATP, alkyl-pyrimidine nucleotides (including propynyl dUTP) are examples of nucleotides having unconventional bases. Some unconventional sugar modifications include modifications at the 2' -position. For example, ribonucleotides with a 2' -OH group (that is, ATP, GTP, CTP, UTP) are unconventional nucleotides of DNA polymerase. Other carbohydrate analogs and modifications include D-ribosyl, 2 'or 3'D-deoxyribosyl, 2',3' -D-dideoxyribosyl, 2',3' -D-didehydrodideoxyribosyl, 2 'or 3' alkoxyribosyl, 2 'or 3' aminoribosyl, 2 'or 3' mercaptoribosyl, 2 'or 3' alkylthioribosyl, acyclic, carbocyclic, or other modified sugar moieties. Further examples include 2' -PO ₄ An analog which is a terminator nucleotide. (see, e.g., U.S. patent No. 7,947,817 or other examples described herein). Unconventional linking nucleotides include phosphorothioate dNTPs ([ alpha-S)]dNTP), 5' - [ alpha-borane]-dNTP and [ alpha ]]-methyl-phosphonate dNTP.

At 214, the selected magnetically-labeled nucleotide precursors are added to the binding region 115.

At 216, sequencing is performed to determine whether the selected magnetically-labeled nucleotide precursor has bound to the polymerase or has incorporated within the extendable primer. As shown in fig. 7, the sequencing step 216 may include a number of sub-steps. For example, at substep 218, one or more lines 120 of the apparatus 100 are used to detect characteristics of the magnetic sensors 105 of the magnetic sensor array 110. As explained above, the characteristic may be, for example, resistance, a change in resistance, a magnetic field, a change in a magnetic field, a frequency, a change in frequency, or noise.

At decision point 220, it is determined whether the detection result indicates that the magnetically labeled nucleotide precursor has been bound to the polymerase or incorporated into the extendable primer. For example, the determination may be based on the presence or absence of a characteristic, e.g., if a characteristic is detected, the magnetically labeled nucleotide precursor is considered to have bound to the polymerase or incorporated into the extendable primer, and if a characteristic is not detected, the magnetically labeled nucleotide precursor is considered to have not bound to the polymerase or incorporated into the extendable primer. As another example, the determination may be based on a magnitude or value of the characteristic, e.g., if the magnitude or value is within a specified range, the magnetically labeled nucleotide precursor is considered to have bound to the polymerase or incorporated into the extendable primer, and if the magnitude or value is not within the specified range, the magnetically labeled nucleotide precursor is considered to have not bound to the polymerase or incorporated into the extendable primer.

The detection (substep 218) and determination (decision point 220) may use or rely on all or less than all of the magnetic sensors 105 in the magnetic sensor array 110. Determining whether a characteristic is present or absent, or a value of the characteristic (decision point 220), may be based on aggregating, averaging, or otherwise processing the detection results from some or all of the magnetic sensors 105 in the magnetic sensor array 110 (substep 218).

If at decision point 220 it is determined that the magnetically-labeled nucleotide precursor has been bound to the polymerase or incorporated into the extendable primer, then at step 222 an indication of the complementary base of the magnetically-labeled nucleotide precursor is recorded in the record of the nucleic acid sequence of the nucleic acid strand.

In some embodiments, the magnetically labeled nucleotide precursor is not extendable by the nucleic acid polymerase and, thus, upon detection of the property, the magnetic label must be removed so that the magnetically labeled nucleotide precursor can be extended by the nucleic acid polymerase. In some embodiments, a portion of the first magnetically-labeled nucleotide precursor is not extendable by a nucleic acid polymerase, and the portion of the first magnetically-labeled nucleotide precursor is extendable by chemical cleavage. As illustrated in fig. 7, if additional sequencing cycles are to be performed ("no" path out of decision point 224), the magnetic labels are removed at 226 using any suitable means (e.g., chemically, enzymatically, or by other means).

After the magnetic label has been removed at 226, another magnetically-labeled nucleotide precursor is selected at 228. Then, at step 214, the newly selected magnetically-labeled nucleotide precursor (which may or may not be the same as the user in the just completed cycle) is added to the binding region 115, and the sequencing step 216 is performed again to determine whether the newly selected magnetically-labeled nucleotide precursor has bound to the polymerase or has incorporated an extendable primer.

If at decision point 220 it is determined that the magnetically labelled nucleotide precursor is not already bound to the polymerase and not incorporated into the extendable primer, the method moves to step 228 where another magnetically labelled nucleotide precursor is selected at said step 228. In this case, because the previously attempted magnetically-labeled nucleotide precursors do not match, the magnetically-labeled nucleotide precursors selected should be different from those used in the just-completed cycle.

Although fig. 7 shows a single optional cleaning step 212 occurring between steps 210 and 214, it should be understood that additional cleaning steps may be included in the method. For example, the binding region(s) 115 may be washed between steps 228 and 214 or after step 226 (e.g., to substantially remove the previously introduced magnetically-labeled nucleotide precursor and any magnetic labels removed in step 226). At 230, method 200 ends.

It will be appreciated that after some number of sequencing cycles, it may be desirable or necessary to perform step 210 to add additional molecules of nucleic acid polymerase to the binding region(s) 115 to replenish the polymerase.

FIG. 7, discussed above, illustrates an embodiment in which magnetically labeled nucleotide precursors are introduced one at a time. In other embodiments, multiple nucleotide precursors (e.g., two, three, or four nucleotide precursors) are introduced substantially simultaneously. In the case of these embodiments, the first and second, different magnetic labels are used for different nucleotide precursors introduced substantially simultaneously. The magnetic labels of the introduced precursors each have a different magnetic property, such that the magnetic sensor 105 can distinguish between the different magnetic labels of the different nucleotide precursors for substantially simultaneous introduction.

FIG. 8 illustrates an embodiment of a method 250 in which multiple nucleotide precursors are introduced to the device 100 substantially simultaneously or another device using a magnetic sensor and magnetic labels for detection. For illustrative purposes, fig. 8 shows four nucleotide precursors introduced substantially simultaneously, but it is understood that the methods disclosed herein can be used to test more or less than four nucleotide precursors.

At 252, method 250 begins. Steps 254, 256, 258, 260 and 262 are the same as steps 204, 206, 208, 210 and 212 shown and described in the context of fig. 7. The description is not repeated here.

At step 264, up to four magnetically labeled nucleotide precursors are added to the binding region(s) 115 of device 100. The added magnetically labeled nucleotide precursors are each labeled with a different magnetic label, such that the magnetic sensor 105 can distinguish between the different magnetically labeled nucleotide precursors. In particular, the magnetic labels each have a distinct and distinguishable magnetic property (e.g., a first magnetic label for a first magnetically-labeled nucleotide precursor has a first magnetic property, a second magnetic label for a second magnetically-labeled nucleotide precursor has a second magnetic property, etc.).

At 266, sequencing is performed to determine which of the added magnetically labeled nucleotide precursors have been bound to the polymerase or incorporated into the extendable primer. As shown in fig. 8, the sequencing step 266 may include a number of sub-steps. For example, in the method 250 set forth in fig. 8, at sub-step 268, one or more lines 120 of the apparatus 100 are used to detect a characteristic of the magnetic sensors 105 of the magnetic sensor array 110, wherein the characteristic identifies the magnetism of the incorporated magnetically-labeled nucleotide precursors. As explained above, the characteristic may be, for example, resistance, a change in resistance, a magnetic field, a change in a magnetic field, a frequency, a change in frequency, or noise.

At decision point 270, it is determined whether a first magnetic property has been detected, wherein the first magnetic property indicates that the first magnetically-labeled nucleotide precursor has bound to the polymerase or has been incorporated into the extendable primer. The determination may be based on, for example, the presence or absence of the first magnetism, e.g., if the first magnetism is detected, the first magnetically-labeled nucleotide precursor is deemed to have bound to the polymerase or incorporated into the extendable primer, and if the first magnetism is not detected, the first magnetically-labeled nucleotide precursor is deemed to have not bound to the polymerase or incorporated into the extendable primer. As another example, the determination may be based on a magnitude or value of the first magnetic property, e.g., if the magnitude or value is within a specified range, the first magnetically labeled nucleotide precursor is deemed to have been bound to the polymerase or incorporated into the extendable primer, and if the magnitude or value is not within the specified range, the first magnetically labeled nucleotide precursor is deemed to have not been bound to the polymerase or incorporated into the extendable primer.

If at decision point 270 it is determined that the first magnetism has been detected, the method moves to step 278 where the complementary base of the first magnetically-labeled nucleotide precursor is recorded in a record of the nucleic acid sequence of the nucleic acid strand.

If at decision point 270 it is determined that the first magnetic property has not been detected, then the method 250 moves to decision point 272 where it is determined whether a second magnetic property has been detected, wherein the second magnetic property indicates that a second magnetically-labeled nucleotide precursor has been bound to the polymerase or incorporated into an extendable primer. The determination may be made in any of the ways described above for determining the first magnetism. If it is determined at decision point 272 that the second magnetic property has been detected, the method moves to step 278 where the complementary base of the second magnetically-labeled nucleotide precursor is recorded in a record of the nucleic acid sequence of the nucleic acid strand.

If at decision point 272, it is determined that the second magnetic property has not been detected, then the method 250 moves to decision point 274, where it is determined whether a third magnetic property has been detected, where the third magnetic property indicates that a third magnetically-labeled nucleotide precursor has been bound to the polymerase or incorporated within an extendable primer. The determination may be made in any of the ways described above for determining the first magnetism. If it is determined at decision point 274 that the third magnetic property has been detected, the method moves to step 278 where the complementary base of a third magnetically-labeled nucleotide precursor is recorded in a record of the nucleic acid sequence of the nucleic acid strand.

Finally, if at decision point 274 it is determined that the third magnetic property has not been detected, then the method 250 moves to decision point 276 where it is determined whether a fourth magnetic property has been detected, wherein the fourth magnetic property indicates that a fourth magnetically-labeled nucleotide precursor has been bound to the polymerase or incorporated into an extendable primer. The determination may be made in any of the ways described above for determining the first magnetism. If it is determined at decision point 276 that the fourth magnetic property has been detected, the method moves to step 278 where the complementary base of the third magnetically-labeled nucleotide precursor is recorded in a record of the nucleic acid sequence of the nucleic acid strand. If at decision point 276 it is determined that the fourth magnetism has not been detected, the method 250 moves back to step 264.

The detection (substep 268) and determination (decision points 270, 272, 274, and 276) may use or rely on all or less than all of the magnetic sensors 105 in the magnetic sensor array 110. Determining whether a particular magnetism is present or absent, or a value of a characteristic, may be based on aggregating, averaging, or otherwise processing detection results from some or all of the magnetic sensors 105 in the magnetic sensor array 110 (substep 268).

In the embodiment illustrated in fig. 8, the determination of which additional magnetically-labeled nucleotide precursors have bound to the polymerase or incorporated into the extendable primer is the result of a "yes/no" determination being made separately for each of the candidate magnetically-labeled nucleotide precursors. It will be appreciated that the decision may alternatively be made in a single step, such as, for example, by comparing the value of the detected characteristic with a key value. For example, the key value may indicate that the first magnetically-labeled nucleotide precursor has bound to the polymerase or incorporated within the extendable primer if the property detected by the magnetic sensor 105 has a value within a first range; if the property detected by the magnetic sensor 105 has a value within a second range, then a second magnetically-labeled nucleotide precursor has bound to the polymerase or incorporated within an extendable primer; if the property detected by the magnetic sensor 105 has a value within a third range, then a third magnetically-labeled nucleotide precursor has bound to the polymerase or incorporated within the extendable primer; and if the property detected by the magnetic sensor 105 has a value within a fourth range, a fourth magnetically-labeled nucleotide precursor has been bound to the polymerase or incorporated within the extendable primer. The values of the characteristics may be based on aggregating, averaging, or otherwise processing the detection results from some or all of the magnetic sensors 105 in the magnetic sensor array 110 (substep 268).

As explained above, in some embodiments, the magnetically-labeled nucleotide precursor cannot be extended by a nucleic acid polymerase, and thus, upon detection of a property, the magnetic label must be removed so that the magnetically-labeled nucleotide precursor can be extended by the nucleic acid polymerase. In some embodiments, a portion of the first magnetically-labeled nucleotide precursor is not extendable by a nucleic acid polymerase, and the portion of the first magnetically-labeled nucleotide precursor is extendable by chemical cleavage. In embodiments where the magnetically-labeled nucleotide precursors cannot be extended by the nucleic acid polymerase, after the recording of the nucleic acid sequence of a nucleic acid strand has been added (or begun) at step 278, it is determined whether additional sequencing cycles are to be performed at decision point 280. If so (the "no" branch of decision point 280), the magnetic label of the incorporated nucleotide precursor is removed. The magnetic labels may be removed chemically, enzymatically, or by other means known in the art, and the method 250 proceeds to step 264, where up to four magnetically labeled nucleotide precursors are added to the binding region 115 (possibly after performing a washing step similar or identical to that described for step 262). Then, sequencing step 266 is performed again to identify the next magnetically labeled nucleotide precursor that binds to the polymerase.

If at decision point 280 it is determined that no additional sequencing cycles are to be performed ("yes" branch of decision point 280), then the method 250 ends at 284.

In the foregoing specification and drawings, specific terminology has been set forth to provide a thorough understanding of the embodiments disclosed herein. In some instances, the terms or figures may imply specific details that are not required to practice the invention.

Well-known elements have been shown in block diagram form and/or have not been discussed in detail, or in some cases, at all, in order to avoid unnecessarily obscuring the present disclosure.

Unless otherwise explicitly defined herein, all terms are given their broadest possible interpretation, including meanings implied by the specification and drawings and meanings understood by those skilled in the art and/or as defined in dictionaries, monographs, etc. Some terms may have a different meaning than their ordinary or customary meaning, as expressly set forth herein.

As used in the specification and the appended claims, the singular forms "a", "an", and "the" do not exclude a plurality of references unless the context clearly dictates otherwise. Unless otherwise specified, the word "or" should be construed as inclusive. Thus, the phrase "a or B" should be construed to mean all of the following: "both A and B", "A but not B" and "B but not A". Any use of "and/or" herein does not imply that the word "or" alone implies exclusivity.

As used in the specification and the appended claims, the phrases "at least one of A, B and C", "at least one of A, B or C", "one or more of A, B or C", and "one or more of A, B and C" are interchangeable and each includes all of the following meanings: all of "a only," B only, "" C only, "" a and B but not C, "" a and C but not B, "" B and C but not a, "and" A, B and C.

To the extent that the terms "includes," including, "" has, "" with, "and variants thereof are used in either the detailed description or the claims, these terms are intended to be inclusive in a manner similar to the term" comprising, "i.e., meaning" including, but not limited to. The terms "exemplary" and "embodiment" are used to mean an example, and not a preference or requirement. The term "coupled" is used herein to mean directly connected/coupled, and connected/coupled through one or more intervening components or structures.

The terms "above … …", "below … …", "between … …" and "on … …" are used herein to refer to the relative position of one feature with respect to another feature. For example, one feature disposed "above" or "below" another feature may be in direct contact with the other feature or may have intervening material. Further, a feature disposed "between" two features may be in direct contact with the two features or may have one or more intervening features or materials. Conversely, a first feature "on" a second feature is in contact with the second feature.

The terms "generally" and "approximately" are used to describe structures, configurations, dimensions, etc., as largely or nearly so specified, but may in practice result in such structures, configurations, dimensions, etc., not always or not necessarily being exactly as specified, due to manufacturing tolerances and the like. For example, describing two lengths as "substantially equal" or "approximately equal" means that for all practical purposes the two lengths are the same, but they may not (and need not) be exactly equal on a sufficiently small scale. As another example, for all practical purposes, a structure that is "substantially vertical" or "nearly vertical" will be considered vertical, even if it is not exactly 90 degrees relative to the horizontal.

The drawings are not necessarily to scale and the size, shape and dimensions of features may be substantially different from the manner in which they are illustrated in the drawings.

Although specific embodiments have been disclosed herein, it will be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the disclosure. For example, features or aspects of any embodiment may be used in combination with or in place of corresponding features or aspects of any other embodiment, at least where practicable. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.

Claims (75)

1. An apparatus for nucleic acid sequencing, the apparatus comprising:

a plurality of magnetic sensors;

a plurality of binding regions disposed above the plurality of magnetic sensors, the binding regions each for containing a fluid; and

at least one line for detecting a characteristic of at least a first magnetic sensor of the plurality of magnetic sensors, the characteristic indicating the presence or absence of one or more magnetic nanoparticles coupled to a first binding region associated with the first magnetic sensor.

2. The device of claim 1, wherein the first magnetic sensor comprises a magnetoresistive device.

3. The apparatus of claim 2, wherein the magneto-resistive device comprises:

a pinning layer;

a free layer; and

a barrier layer configured between the pinned layer and the free layer.

4. The apparatus of claim 3, wherein, in the absence of the one or more magnetic nanoparticles coupled to the first binding region, the magnetic moment of the pinned layer is about 90 degrees from the magnetic moment of the free layer.

5. The device of any of claims 1-4, wherein the first magnetic sensor is substantially cylindrical or substantially cubical in shape.

6. The device of any of claims 1-5, wherein a lateral dimension of the first magnetic sensor is between about 10 nanometers and about 1 micron.

7. The device of any one of claims 1-6, further comprising sensing circuitry coupled to the plurality of magnetic sensors via the at least one pipeline, wherein the sensing circuitry is configured to:

applying a current to the at least one line to detect the characteristic of the first magnetic sensor.

8. The device of any of claims 1-7, wherein the characteristic includes a magnetic field or a resistance.

9. The device of any one of claims 1-8, wherein the characteristic includes a change in a magnetic field or a change in resistance.

10. The device of any of claims 7-9, wherein the sensing circuitry includes a magnetically controlled oscillator, and wherein the characteristic includes a frequency of a signal associated with or generated by the magnetically controlled oscillator.

11. The device of any one of claims 1-10, wherein the characteristic includes a noise level.

12. The device of any one of claims 1-11, further comprising an insulating material configured between the plurality of magnetic sensors and the plurality of binding regions.

13. The device of claim 12, wherein the insulating material comprises at least one of silicon dioxide, aluminum oxide, or silicon nitride.

14. The device of claim 12 or claim 13, wherein the insulating material comprises at least one of an oxide or a nitride.

15. The device of any of claims 12-14, wherein a thickness of the insulating material between a top of the first magnetic sensor and the first binding region is between about 3 nanometers and about 20 nanometers.

16. The device of any one of claims 1-15, wherein the at least one conduit comprises a first conduit configured above a top surface of the first magnetic sensor, and wherein the first binding region is located within a trench in the first conduit, the trench being above the top surface of the first magnetic sensor.

17. The device of any one of claims 1-16, wherein the plurality of magnetic sensors are disposed in a rectangular array, and wherein the at least one conduit comprises at least a first conduit and a second conduit, wherein the first conduit is configured above the first magnetic sensor and the second conduit is configured below the first magnetic sensor.

18. The apparatus of claim 17, wherein the first bonding region is located within a trench in the first pipeline.

19. The device of claim 17 or claim 18, wherein the first pipeline is a row coupled to the rectangular array and the second pipeline is a column coupled to the rectangular array, or vice versa.

20. The device of any one of claims 1-19, wherein the first binding region comprises a structure configured to anchor a nucleic acid to the first binding region.

21. The device of claim 20, wherein the structure comprises a cavity or a ridge.

22. A method of sequencing nucleic acids using a device comprising a plurality of magnetic sensors; a plurality of binding regions disposed above the plurality of magnetic sensors, the binding regions each for containing a fluid; and at least one conduit for detecting a characteristic of at least a first magnetic sensor of the plurality of magnetic sensors, the method comprising:

(a) Binding at least one nucleic acid strand to the first binding region;

(b) Adding an extendable primer and a nucleic acid polymerase to the first binding region in one or more rounds of addition;

(c) Adding a first nucleotide precursor to the first binding region, the first nucleotide precursor labeled with a first cleavable magnetic label; and

(d) Sequencing the nucleic acid strands,

wherein sequencing the nucleic acid strand comprises:

using the at least one pipeline, detecting the characteristic of the first magnetic sensor indicative of the presence or absence of the first cleavable magnetic label.

23. The method of claim 22, further comprising amplifying the at least one nucleic acid strand.

24. The method of claim 22 or claim 23, further comprising amplifying the at least one nucleic acid strand after binding the at least one nucleic acid strand to the first binding region.

25. The method of claim 23 or claim 24, wherein one or more amplicons are bound to the first binding region as a result of the amplification.

26. The method of any one of claims 22-25, wherein sequencing the nucleic acid strands further comprises:

in response to a determination that the property indicates the presence of one or more magnetic nanoparticles coupled to the first binding region, recording a complementary base of the first nucleotide precursor in a recording of a nucleic acid sequence of the nucleic acid strand.

27. The method of any one of claims 22-26, wherein the first nucleotide precursor is not extendable by the nucleic acid polymerase, and further comprising:

after detecting the property, removing the first cleavable magnetic label and enabling extension of the first nucleotide precursor by the nucleic acid polymerase.

28. The method of any one of claims 22-26, wherein the first nucleotide precursor is not extendable by the nucleic acid polymerase.

29. The method of claim 28, wherein the first nucleotide precursor is rendered extendable by chemical cleavage.

30. The method of any one of claims 22-29, further comprising removing the cleavable magnetic labels by enzymatic or chemical cleavage after sequencing the nucleic acid strands.

31. The method of any one of claims 22-30, further comprising:

during each repetition, steps (c) and (d) are repeated with different nucleotide precursors, each of which is magnetically labeled.

32. The method of claim 31, wherein the first and different nucleotide precursors are each selected from magnetically labeled adenine, guanine, cytosine, thymine, or equivalents thereof.

33. The method of any one of claims 22-32, further comprising, prior to step (c), washing the first bonding area.

34. The method of any one of claims 22-33, wherein the first cleavable magnetic label has a first magnetic property, and wherein the method further comprises:

in the one or more additions, a second nucleotide precursor labeled with a second cleavable magnetic label having a second magnetic property is added to the first binding region.

35. The method of claim 34, further comprising:

in the one or more additions, a third nucleotide precursor labeled with a third cleavable magnetic label having a third magnetic property and a fourth nucleotide precursor labeled with a fourth cleavable magnetic label having a fourth magnetic property are added to the first binding region.

36. The method of any one of claims 22-35, wherein binding the at least one nucleic acid strand to the first binding region comprises:

binding an adapter to an end of a respective one of the at least one nucleic acid strand; and

coupling an oligonucleotide to the first binding region, wherein the oligonucleotide is capable of hybridizing to the adapter.

37. The method of any one of claims 22-36, wherein binding the at least one nucleic acid strand to the first binding region comprises covalently binding each of the at least one nucleic acid strands to the first binding region.

38. The method of any one of claims 22-37, wherein binding the at least one nucleic acid strand to the first binding region comprises immobilizing the at least one nucleic acid strand via irreversible passive adsorption or intermolecular affinity.

39. The method of any one of claims 22-38, wherein the first binding region comprises a cavity or a ridge, and wherein binding the at least one nucleic acid strand to the first binding region comprises applying a hydrogel to the cavity or to the ridge.

40. The method of any one of claims 22-39, further comprising:

after step (c), adding an additional molecule of the nucleic acid polymerase to the first binding region.

41. The method of any one of claims 22-40, wherein the first cleavable magnetic label comprises a magnetic nanoparticle.

42. The method of claim 41, wherein the magnetic nanoparticles are molecules.

43. The method according to claim 41, wherein the magnetic nanoparticles are superparamagnetic nanoparticles.

44. The method of claim 41, wherein the magnetic nanoparticles are ferromagnetic nanoparticles.

45. The method of any one of claims 22-44, wherein the first nucleotide precursor comprises one of dATP, dGTP, dCTP, dTTP, or an equivalent.

46. The method of any one of claims 22-45, wherein the nucleic acid polymerase comprises a type B polymerase lacking 3'-5' exonuclease activity.

47. The method of any one of claims 22-46, wherein the nucleic acid polymerase comprises a thermostable polymerase.

48. The method of any one of claims 22-47, wherein using the at least one line comprises applying a current to the at least one line.

49. The method of any one of claims 22-48, wherein the characteristic comprises a magnetic field or an electrical resistance.

50. The method of any one of claims 22-49, wherein the characteristic includes a frequency of a signal associated with or generated by a magnetically controlled oscillator.

51. The method of any one of claims 22-50, wherein the characteristic includes a noise level.

52. The method of any one of claims 22-51, wherein the characteristic includes a change in magnetic field or a change in resistance.

53. The method of any one of claims 22-52, wherein the characteristic results from a change in magnetic field or a change in resistance.

54. A method of fabricating a nucleic acid sequencing device, the method comprising:

manufacturing a first pipeline;

fabricating a plurality of magnetic sensors, each magnetic sensor having a bottom surface and a top surface, wherein each bottom surface is coupled to the first pipeline;

depositing an insulating material between the magnetic sensors;

fabricating a plurality of additional conduits, each coupled to the top surface of a respective magnetic sensor of the plurality of magnetic sensors; and

a plurality of binding domains is generated.

55. The method of claim 54, wherein fabricating the first pipeline comprises:

depositing a metal layer on a substrate; and

patterning the metal layer into the first pipeline.

56. The method of claim 55, wherein depositing the metal layer on the substrate comprises depositing the metal layer using physical vapor deposition or ion beam deposition.

57. The method of claim 55 or claim 56, wherein patterning the metal layer into a first pipeline comprises patterning the metal layer using one or more of photolithography, milling, or etching.

58. The method of any one of claims 54-57, further comprising, after fabricating the first pipeline and before fabricating the plurality of magnetic sensors:

depositing an insulating material over the first pipeline; and

the first pipeline is exposed to the outside and,

and wherein fabricating the plurality of magnetic sensors comprises fabricating the plurality of magnetic sensors on the bare first pipeline.

59. The method of claim 58, wherein exposing the first line comprises using Chemical Mechanical Polishing (CMP).

60. The method of any one of claims 54 to 59, wherein fabricating the plurality of magnetic sensors comprises:

depositing a plurality of layers on the first pipeline; and

patterning the plurality of layers to form the plurality of magnetic sensors, the plurality of magnetic sensors each have a predetermined shape.

61. The method of claim 60, wherein depositing the plurality of layers comprises:

depositing a first ferromagnetic layer;

depositing a metal or insulating layer over the first ferromagnetic layer; and

a second ferromagnetic layer is deposited over the metal or insulating layer.

62. The method of claim 60 or claim 61, wherein patterning the plurality of layers to form the plurality of magnetic sensors comprises at least one of photolithography or etching.

63. The method of any one of claims 60 to 62, wherein the predetermined shape is substantially cylindrical or substantially cubical.

64. The method according to any one of claims 54-63, wherein a lateral dimension of each of the plurality of magnetic sensors is between about 10 nanometers and about 1 micrometer.

65. The method of any one of claims 54-64, wherein:

fabricating the plurality of magnetic sensors includes fabricating the plurality of magnetic sensors in a rectangular array,

and wherein the first pipeline corresponds to a row of the rectangular array and the plurality of further pipelines each correspond to a column of the rectangular array.

66. The method of any one of claims 54-64, wherein:

fabricating the plurality of magnetic sensors includes fabricating the plurality of magnetic sensors in a rectangular array,

and wherein the first pipeline corresponds to a column of the rectangular array and the plurality of further pipelines each correspond to a row of the rectangular array.

67. The method of any one of claims 54 to 66, further comprising, after depositing the insulating material between the magnetic sensors and prior to fabricating the plurality of additional lines:

a chemical mechanical polishing step is performed to expose the top surface of each of the plurality of magnetic sensors.

68. The method of any one of claims 54-67, wherein fabricating the plurality of additional lines comprises:

depositing a metal layer;

performing photolithography to define the plurality of further lines; and

removing a portion of the metal layer.

69. The method of any one of claims 54-68, wherein generating the plurality of binding regions comprises:

applying a mask over the plurality of bonding regions;

depositing a metal layer over the mask; and

and picking up the mask.

70. The method of claim 69, further comprising:

after the mask is extracted, additional insulating material is deposited over the plurality of additional lines and the plurality of bonding regions.

71. The method of claim 70, wherein the thickness of the additional insulating material is between about 3 nanometers and about 20 nanometers.

72. The method of claim 70 or claim 71, wherein the additional insulating material comprises an oxide or a nitride.

73. The method of any one of claims 70-72, wherein the additional insulating material comprises silicon dioxide (SiO) ₂ )。

74. The method of any one of claims 70-73, wherein depositing comprises performing atomic layer deposition.

75. The method of any one of claims 54 to 74, wherein fabricating comprises depositing.

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